Immunocytochemistry/ Immunofluorescence abreview for Anti-Ki67 antibody [SP6] - Proliferation Marker

Excellent
Abreviews
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEp-2)
Specification
HEp-2
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100 0.25% in PBS

Other product details

Dilution
1/50
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 21°C · Diluent: DPBS

Secondary antibody

Name
Non-Abcam antibody was used: AffiniPurre F(ab')2 Fragment Donkey Anti-Rabbit Ig
Host species: Donkey
Clonality: Polyclonal
Conjugation: Other
Dilution
1/50

Additional data

Additional Notes
Immunostaining protocol:
Cells cultured on Coverslips were rinsed with Dulbeccos phosphate-buffered saline with Calcium and Magnesium (DPBS).
The sample was fixed in 4% paraformaldehyde  in PBS pH 7.4 (room temperature) for 10 minutes and permeabilized by incubation in Triton X-100 (0,25% v/v in DPBS) for 5 min.
Cells were washed in DPBS three times for 5 min.
Cells were incubated in the primary antibody solution diluted 1:50 in DPBS in a humidified chamber for 1 hr at room temperature. Then cells were washed three times in DPBS, 5 min each wash.
Cells were incubated with the secondary antibody (dilution 1:50) in DPBS for 1 hr at room temperature in dark and finally washed three times in DPBS again.

Additional remarks:
Cells were stained without any blocking before or during antibody incubation. Since only very low background signal was observed, no protocols including a blocking step were tested. However, further reduction of unspecific signal might be possible.

Secondary Antibody (IgG, F(ab)2-fragment, donkey anti-rabbit IgG (H+L), unconj., with minimal cross-reactivity to Bo, Ck, Go, Gp, Hs, Ho, Hu, Ms, Rt, Sh dianova GmbH, Hamburg, Germany) was labelled with Atto488 using Atto488-NHS-Ester (ATTO-Tec GmbH, Siegen, Germany) in a dilution of 1:50.

Mr. Peter Zentis

Verified customer

Submitted Sep 10 2010

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